Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative commensal of the human gingival crevice and is an opportunistic periodontal pathogen. The gingival crevice is a microbe-rich environment, and Aa responds to cues from its neighbors, such as Streptococcus gordonii (Sg), altering its diet [3] and avoiding host immune defenses [4]. While these interactions are thought to be important for the survival of Aa in the oral cavity, the genetic regulatory mechanisms mediating these interspecies interactions are largely unknown. Nearly all bacteria utilize non-coding RNAs, including small non-coding regulatory RNAs (sRNAs) and metabolite sensing riboswitches, to alter gene expression but none have been characterized in Aa. This led to the hypothesis that Aa expresses regulatory RNAs to alter gene expression in response to changes in the environment and cues from neighboring bacteria. Initially, Northern blot analysis was used to detect the expression of 23 predicted sRNAs (http://www.oralgen.lanl.gov/). 12 differentially expressed putative sRNAs were detected. I have characterized the first lysine riboswitch and the novel lysine transporter, LysT, in Aa. Since the riboswitch has a long half-life and is expressed at high levels, I hypothesize that the lysine riboswitch has another role in the cell as a trans-acting sRNA. In the first aim I propose experiments to determine trans regulatory targets for the lysine riboswitch. I will also identify lysine-dependent changes in gene expression. The second aim of this proposal builds off of the first aim, but takes a global approach to identify regulatory RNAs in a polymicrobial biofilm community with Aa and Sg. This technology and training based approach will use next generation deep sequencing to map transcriptional start sites and transcripts from Aa and Sg, as well as identify differentially regulated transcripts and candidate regulatory RNAs expressed by both bacteria, including sRNAs, riboswitches, and antisense RNAs. This study will provide the first multispecies transcriptional regulome; identifying regulatory RNAs expressed by two bacteria in a polymicrobial community, and will provide important information for the oral microbiology research community that will lead to future studies of genetic regulation in both Aa and Sg. Throughout the proposed work, techniques will be learned in bioinformatics, biochemistry, genetics, and molecular biology, and will provide an excellent foundation for further research endeavors. PUBLIC HEALTH RELEVANCE: Interactions between multiple species of bacteria are important for colonization of oral tissues and bacterial defenses to host immunity. The goal of this proposal is to characterize the regulatory mechanisms mediating bacterial multispecies interactions in the mouth, and develop new technology that will provide powerful tools for the research community. These studies will help us better understand how oral bacteria thrive in polymicrobial communities that cause human disease.